lncRNA SNHG1 Knockdown Alleviates Amyloid-β-Induced Neuronal Injury by Regulating ZNF217 via Sponging miR-361-3p in Alzheimer’s Disease

Abstract

Background:

Long noncoding RNAs have been proven to play an important role in the progression of Alzheimer’s disease (AD). However, the function of small nucleolar RNA host gene 1 (SNHG1) in AD progression remains to be studied.

Objective:

To explore the role of SNHG1 in AD progression and clarify its potential mechanism.

Methods:

Amyloid β-protein (Aβ) was used to construct an AD cell model in vitro. The expression levels of SNHG1 and miR-361-3p were determined by quantitative real-time polymerase chain reaction. Cell viability and apoptosis were measured by cell counting kit 8 assay and flow cytometry. The levels of apoptosis-related proteins and zinc finger gene 217 (ZNF217) protein were evaluated by western blot analysis. Additionally, the contents of inflammatory cytokines and oxidative stress markers were tested by enzyme-linked immunosorbent assay. Furthermore, dual-luciferase reporter and RNA immunoprecipitation assays were used to verify the interaction between miR-361-3p and SNHG1 or ZNF217.

Results:

Aβ could induce cell injury, while resveratrol could reverse this effect. SNHG1 expression was positively regulated by Aβ and negatively regulated by resveratrol. SNHG1 knockdown could reverse the promotion effect of Aβ on cell injury. Moreover, SNHG1 sponged miR-361-3p, and miR-361-3p targeted ZNF217. Additionally, miR-361-3p overexpression reversed the promotion effect of SNHG1 overexpression on cell injury, and ZNF217 silencing also reversed the promotion effect of miR-361-3p inhibitor on cell injury.

Conclusion:

SNHG1 promoted cell injury by regulating the miR-361-3p/ZNF217 axis, which might provide a theoretical basis for molecular therapy of AD.

Keywords

Alzheimer’s disease amyloid-β miR-361-3p resveratrol SNHG1 ZNF217

INTRODUCTION

Alzheimer’s disease (AD) is a progressive degenerative disease of the nervous system characterized by a decline or loss of cognitive and memory abilities [1, 2]. There are many pathogenic mechanisms for AD, including increased inflammation, death of dying neurons, and brain shrinkage [3]. Amyloid-β protein (Aβ) has a strong neurotoxic effect, and its excessive deposition is considered to be a key clinicopathological factor in the formation of AD [4]. Currently, there is no cure for AD, but medication and behavior modification can slow the progression of AD [5]. Resveratrol (Res) is a polyphenolic compound with strong antioxidant properties [6]. Several clinical trials have shown that Res reduces nerve inflammation, relieves nerve injury, and thus alleviate AD progression [7, 8]. Therefore, its neuroprotective effect can also help us screen molecular markers for early diagnosis of AD.

Long noncoding RNAs (lncRNAs) are long-stranded RNA molecules of more than 200 nucleotides in length [9]. In recent years, researchers have found that lncRNA is also involved in the regulation of AD progression [10]. For example, lncRNA MALAT1 could suppress neuron apoptosis, inflammation, and enhance neurite outgrowth in AD [11]. Besides, knockdown of lncRNA NEAT1 alleviated the neuronal injury induced by Aβ [12], and overexpression of lncRNA MEG3 could improve AD cognitive dysfunction and reduce neuron injury [13]. Small nucleolar RNA host gene 1 (SNHG1) is a lncRNA that is widely higher expressed in a variety of diseases and is considered to be an oncogene regulating cancer progression [14, 15]. Qian et al. reported that silenced SNHG1 could promote neuronal autophagy and suppress cell death in Parkinson’s disease [16]. Additionally, Wang et al. reported that SNHG1 knockdown could attenuate Aβ-induced neuronal injury, which was mainly achieved by regulating cell viability and apoptosis [17]. Therefore, exploring the role of SNHG1 in AD might provide new ideas for alleviating the progression of AD.

There are many mechanisms of lncRNAs, one of which is that as a sponge of microRNA (miRNA) to participate in the regulation of downstream genes [18]. miR-361-3p had been proven to be highly expressed in AD and could inhibit Aβ accumulation, thereby relieving the cognitive impairment of AD patients [19]. However, its mechanism in AD was not yet perfect. Besides, zinc finger gene 217 (ZNF217) has been shown to be a potential oncogene in many cancers [20]. Moreover, studies had suggested that the low expression of ZNF217 could alleviate Aβ-induced neurotoxicity in AD [21].

The purpose of this study was to explore the role of SNHG1 in Aβ-induced neuronal injury and Res-treated neuronal repair. According to the hypothesis of lncRNA/miRNA/mRNA axis, we further investigated the mechanism of SNHG1 based on bioinformatics analysis and experimental verification. Our results might provide a reliable theoretical basis for the treatment of AD.

MATERIALS AND METHODS

Cell culture and treatment

Neuroblastoma cell line (SK-N-SH and CHP 212) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (100 U/mL–100μg/mL) (Solarbio, Beijing, China) at 37°C in a 5% CO₂ incubator. Aβ (Aβ_25–35; Meilinbio, Dalian, China) and Res (Meilinbio) were added to SK-N-SH and CHP 212 cells every time the medium was changed and continuous treatment until cells were collected.

Cell counting kit 8 (CCK8) assay

According to the procedures of the previous study [15], SK-N-SH and CHP 212 cells were seeded into 96-well plates and incubated for 48 h. After that, the cells were incubated with 10μL CCK8 (Amyjet, Wuhan, China) for 2 h. Then, the absorbance was measured to evaluate the viability of cells using a microplate reader (Mindray, Shenzhen, China) at 450 nm.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNAs were harvested using TRIzol reagent (Invitrogen, Waltham, MA, USA) and reverse-transcribed into cDNA using a First-Strand cDNA Synthesis Kit (GeneCopoeia, Rockville, MD, USA). qRT-PCR was performed using SYBR Premix (Takara, Dalian, China). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were used as internal controls. The relative gene expression was calculated using the 2^- ΔΔCt method. The primer sequences were as follows: SNHG1, F 5’-ACGTTGGAACCGAAGAGAGC-3’, R 5’-GCAGCTGAATTCCCCAGGAT-3’; GAPDH, F 5’-GAGTCCACTGGCGTCTTCAC-3’, R 5’-ATCTTGAGGCTGTTGTCATACTTCT-3’; miR-361-3p, F 5’-UCCCCCAGGUGUGAUUCUGAUUU-3’, R 5’-GCAAATCAGAATCACACCTG-3’; U6, F 5’-CTCGCTTCGGCAGCACA-3’, R 5’-AACGCTTCACGAATTTGCGT-3’. All primers were designed by us using the NCBI software and synthesized by Invitrogen.

Flow cytometry

The procedures were consistent with previous research [17]. Briefly, SK-N-SH and CHP212 cells were digested by trypsin and the cell suspension was collected after treatment or transfection, Then, cell suspension was stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (Vazyme, Nanjing, China) for 30 min. Fluorescence signals were assessed and the apoptosis of cells was calculated by the Flow cytometer (Beckman Coulter, San Jose, CA, USA).

Western blot (WB) analysis

Total proteins were extracted using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) and quantified using bicinchoninic acid (BCA) Kit (Beyotime) according to previous studies [16]. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocked with 5% non-fat milk for 1 h, the membranes were incubated with the following primary antibodies: B-cell lymphoma-2 (Bcl-2, 1 : 750, Beyotime), Bcl2-associated X (Bax, 1 : 1,000, Beyotime), caspase 3 (1 : 1,000, Beyotime), ZNF217 (1 : 2,000, Invitrogen), or GAPDH (1 : 1,000, Beyotime) at 4°C overnight. After incubated with secondary antibody (1 : 2,000, Beyotime) for 2 h, the membranes were visualized using an enhanced chemiluminescence solution (Beyotime) to detect the protein signals of cells.

Enzyme-linked immunosorbent assay (ELISA)

After induction or transfection, the culture mediums of SK-N-SH and CHP 212 cells were collected into a centrifuge tube, and the supernatant was taken for test after centrifugation. The contents of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, superoxide dismutase (SOD), and malondialdehyde (MDA) were measured by their corresponding ELISA Kits (MSK Bio, Wuhan, China) according to the manufacture’s instruction.

Cell transfection

SNHG1 small interfering RNA (siRNA) and overexpression plasmid (si-SNHG1#1/2/3 and SNHG1) or their negative controls (si-NC and lnc-NC), miR-361-3p mimic and inhibitor (miR-361-3p and anti-miR-361-3p) or their negative controls (NC and anti-NC), ZNF217 siRNA (si-ZNF217) and its negative control (scramble) were synthesized by Ribobio (Guangzhou, China). When cell reached 60–70% confluence, cell transfection could be carried out. Lipofectamine 3000 (Invitrogen) was performed to transfect all plasmid vectors into SK-N-SH and CHP 212 cells according to the manufacturer’s instructions.

Dual-luciferase reporter assay

Similarly with the previous studies [12], the sequences of SNHG1 containing predicted miR-361-3p binding sites or mutant binding sites were inserted into the pmirGLO vectors (Promega, Madison, WI, USA) to build the SNHG1 wild type or mutant type (SNHG1-wt or SNHG1-mut) reporter vector. Similarly, ZNF217-wt or ZNF217-mut reporter vector was constructed in the same way. The above reporter vectors were co-transfected with miR-361-3p mimic or NC into SK-N-SH and CHP 212 cells using Lipofectamine 3000 (Invitrogen). Then, Dual-Lucy Assay Kit (Solarbio, Beijing, China) was used to determine the luciferase activities of cells.

RNA immunoprecipitation (RIP) assay

SK-N-SH and CHP 212 cells were lysed with RIP lysis buffer (Millipore) and incubated with immunoglobulin G (IgG) antibody (Anti-IgG) or argonaute2 (Ago2) antibody (Anti-Ago2) coated on magnetic beads (Millipore) at 4°C overnight. After RNAs were purified from the immunoprecipitates using TRIzol reagent, the enrichment of SNHG1, miR-361-3p and ZNF217 were detected by qRT-PCR.

Statistical analysis

Data were presented as the mean±standard deviation. Student’s t-test was used to compare the difference between two groups, and two-way analysis of variance (ANOVA) followed by Tukey post hoc test was used to compare the differences among multiple groups. GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA) was used to analyze the data. p < 0.05 was defined as statistically significant.

RESULTS

Res could alleviate the effect of Aβ on the viability and SNHG1 expression of SK-N-SH and CHP 212 cells

Firstly, in order to determine the effect of Aβ on cells, we treated SK-N-SH and CHP 212 cells with different concentrations of Aβ and tested cell viability. The results showed that with the increase of Aβ concentration, cell viability was significantly decreased (Fig. 1A). Then, 20μM concentration of Aβ and different concentrations of Res were used to treat SK-N-SH and CHP 212 cells. As shown in Fig. 1B, we found that the viability of cells was increased after Res treatment in a concentration-dependent manner, which indicated that Res had a restorative effect on Aβ-inhibited cell viability. Interestingly, we discovered that the level of SNHG1 was changed, which was remarkably increased after Aβ treatment and markedly decreased after Res treatment in SK-N-SH and CHP 212 cells (Fig. 1C), indicating that the expression of SNHG1 might be related to cell injury.

Fig. 1

Effects of Res and Aβ on the viability and SNHG1 expression of SK-N-SH and CHP 212 cells. A) CCK8 assay was used to measure the viability of SK-N-SH and CHP 212 cells treated with different concentrations of Aβ. B) The viability of SK-N-SH and CHP 212 cells treated with 20μM Aβ and different concentrations of Res was detected by CCK8 assay. C) The expression of SNHG1 was assessed by qRT-PCR in SK-N-SH and CHP 212 cells treated with 20μM Aβ and 20 nM Res. Student’s t-test was used for comparison. *p < 0.05.

Res could reverse the effect of Aβ on the viability, apoptosis, inflammatory response, and oxidative stress of SK-N-SH and CHP 212 cells

To further understand the effect of Res on Aβ-induced cell injury, we treated SK-N-SH and CHP 212 cells with 20μM Aβ and 20 nM Res. The detection of CCK8 assay showed that Res could recover the suppression effect of Aβ on the viability of SK-N-SH and CHP 212 cells (Fig. 2A). Flow cytometry results revealed that cell apoptosis was markedly enhanced after Aβ treated, while Res could invert this promotion effect (Fig. 2B). Besides, by measuring the levels of apoptosis-related protein, we found that Res could also reverse the promoting effect of Aβ on Bax and Cleaved caspase 3/pro-caspase 3 levels and the inhibiting effect of it on Bcl-2 level in SK-N-SH and CHP 212 cells (Fig. 2C, D). Furthermore, the detection results of inflammatory cytokine TNF-α, IL-1β, and IL-6 levels showed that Aβ promoted the inflammatory response of SK-N-SH and CHP 212 cells, and this promotion effect could be inverted by Res (Fig. 2E–G). Meanwhile, we also assessed the contents of oxidative stress marker SOD and MDA and found that Aβ could inhibit the SOD level and increase the MDA level, suggesting that it promoted the oxidative stress of SK-N-SH and CHP 212 cells, while Res could invert this effect (Fig. 2H–I). These results suggested that Res could effectively alleviate cell injury induced by Aβ.

Fig. 2

Effects of Res and Aβ on SK-N-SH and CHP 212 cell injury. SK-N-SH and CHP 212 cells were treated with Aβ and Res. A) CCK8 assay was performed to assess the viability of SK-N-SH and CHP 212 cells. B) The apoptosis of SK-N-SH and CHP 212 cells was measured by flow cytometry. C, D) The protein levels of Bax, Bcl-2 and Cleaved caspase 3/pro-caspase 3 in SK-N-SH and CHP 212 cells were detected by WB analysis. E-I) The contents of TNF-α, IL-1β, IL-6, SOD, and MDA in SK-N-SH and CHP 212 cells were assessed by ELISA assay. Student’s t-test was used for comparison in A, and two-way ANOVA followed by Tukey post hoc test was used for comparison in B–I. *p < 0.05.

Knockdown of SNHG1 could invert the effect of Aβ on the viability, apoptosis, inflammatory response, and oxidative stress of SK-N-SH and CHP 212 cells

In view of the increased expression of SNHG1 during Aβ treatment of SK-N-SH and CHP 212 cells, we constructed si-SNHG1 to further explore the influence of SNHG1 expression on cell injury induced by Aβ. QRT-PCR results showed that si-SNHG1 had a good inhibitory effect on the expression of SNHG1 (especially si-SNHG1#1), so it could be used for subsequent experiments (Fig. 3A). We transfected si-SHNH1#1 into Aβ-treated SK-N-SH and CHP 212 cells to assess the role of SNHG1 on cell injury. The detection results of SNHG1 expression indicated that SHNG1 could effectively reverse the promoting effect of Aβ on SNHG1 expression in SK-N-SH and CHP 212 cells (Fig. 3B). Subsequently, we measured the viability, apoptosis, inflammatory response, and oxidative stress of SK-N-SH and CHP 212 cells. CCK8 assay results revealed that SNHG1 silencing reversed the inhibition effect of Aβ on the viability of SK-N-SH and CHP 212 cells (Fig. 3C), and flow cytometry results indicated that the promotion effect of Aβ on apoptosis also could be inverted by SNHG1 knockdown (Fig. 3D). At the same time, the decrease of Bax and cleaved caspase 3/pro-caspase 3 expression and the increase of Bcl-2 expression also confirmed the recovery effect of SNHG1 knockdown on Aβ-induced apoptosis in SK-N-SH and CHP 212 cells (Fig. 3E, F). Besides, we also discovered that silenced SNHG1 could reverse the promotion effect of Aβ on the inflammatory response and oxidative stress of SK-N-SH and CHP 212 cells, as demonstrated by detecting the TNF-α, IL-1β, IL-6, SOD, and MDA contents (Fig. 3G–K). Therefore, all data revealed that SNHG1 knockdown might be an effective method to alleviate Aβ-induced cell injury.

Fig. 3

The function of SNHG1 silencing on Aβ-induced SK-N-SH and CHP 212 cell injury. A) The expression of SNHG1 was detected by qRT-PCR to evaluate the transfection efficiency of si-SNHG1#1/2/3. SK-N-SH and CHP 212 cells were treated with 20μM Aβ and transfected with si-SNHG1#1 or si-NC. B) QRT-PCR was performed to test the SNHG1 expression in SK-N-SH and CHP 212 cells. C) The viability of SK-N-SH and CHP 212 cells was measured by CCK8 assay. D) The apoptosis of SK-N-SH and CHP 212 cells was assessed by flow cytometry. E, F) WB analysis was used to determine the protein levels of Bax, Bcl-2, and Cleaved caspase 3/pro-caspase 3 in SK-N-SH and CHP 212 cells. G–K) ELISA assay was performed to test the contents of TNF-α, IL-1β, IL-6, SOD, and MDA in SK-N-SH and CHP 212 cells. Two-way ANOVA followed by Tukey post hoc test was used for comparison. *p< 0.05.

MiR-361-3p could be targeted by SNHG1

To explore the mechanism of SNHG1 as a miRNA sponge, we predicted the potential targeted miRNAs of SNHG1 using the ENCORI tool. As shown in Fig. 4A, miR-361-3p was found to have complementary sites with SNHG1. To further confirm the binding ability between miR-361-3p and SNHG1, we constructed SNHG1-wt and SNHG1-mut reporter vectors to perform the dual-luciferase reporter assay. The results determined that miR-361-3p overexpression could remarkably inhibit the luciferase activity of SNHG1-wt in SK-N-SH and CHP 212 cells, but no effect on SNHG1-mut (Fig. 4B, C). Meanwhile, RIP assay results suggested that SNHG1 and miR-361-3p in SK-N-SH and CHP 212 cells were markedly enriched in Anti-Ago2 (Fig. 4D). To investigate the expression of SNHG1 on miR-361-3p, we built SNHG1 overexpression plasmid. QRT-PCR results showed that SNHG1 overexpression plasmid had a good promotion effect on the expression of SNHG1, indicating a good transfection efficiency (Fig. 4E). Next, we transfected SNHG1 overexpression plasmid and si-SNHG1#1 into SK-N-SH and CHP 212 cells to detect the expression of miR-361-3p. The results uncovered that miR-361-3p expression was suppressed by SNHG1 overexpression and enhanced by SNHG1 knockdown in SK-N-SH and CHP 212 cells, indicating that miR-361-3p expression was regulated by SNHG1 (Fig. 4F). Hence, the above results confirmed that miR-361-3p was targeted by SNHG1 in SK-N-SH and CHP 212 cells.

Fig. 4

MiR-361-3p could be targeted by SNHG1. A) The sequences of SNHG1 containing the miR-361-3p binding sites or mutant binding sites were shown. B, C) Dual-luciferase reporter assay was used to detect the interaction between miR-361-3p and SNHG1 in SK-N-SH and CHP 212 cells. D) RIP assay was performed to determine the enrichment of SNHG1 and miR-361-3p in Anti-Ago2 or Anti-IgG. E) The expression of SNHG1 was measured by qRT-PCR to evaluate the transfection efficiency of SNHG1 overexpression plasmid. F) QRT-PCR was performed to test the expression of miR-361-3p in SK-N-SH and CHP 212 cells to assess the effect of SNHG1 expression on miR-361-3p expression. Student’s t-test was used for comparison. *p < 0.05.

Effects of SNHG1 overexpression and miR-361-3p mimic on the treatment of Aβ and Res in SK-N-SH and CHP 212 cells

To further explore the role of miR-361-3p, we first assessed the transfection efficiency of miR-361-3p mimic. QRT-PCR results showed that miR-361-3p mimic had a good promotion effect on the expression of miR-361-3p, suggesting a good transfection efficiency (Fig. 5A). Therefore, we co-transfected SNHG1 overexpression plasmid and miR-361-3p mimic into Aβ and Res treated SK-N-SH and CHP 212 cells. Through the detection of the miR-361-3p expression, we found that Res reversed the inhibition effect of Aβ on miR-361-3p expression, and miR-361-3p mimic could invert the suppression effect of SNHG1 overexpression on miR-361-3p expression (Fig. 5B). Also, the measurement results of viability and apoptosis abilities indicated that SNHG1 overexpression could reverse the promoting effect of Res on viability and the inhibiting effect of it on apoptosis in Aβ-induced SK-N-SH and CHP 212 cells, while overexpressed miR-361-3p also could invert the functions of SNHG1 overexpression on cell viability and apoptosis (Fig. 5C, D). Similarly, the decreasing effect of Res on the Bax and Cleaved caspase 3/pro-caspase 3 protein levels and the increasing effect of it on the Bcl-2 protein level could be recovered by the ectopic expression of SNHG1, whereas the miR-361-3p overexpression reversed the effect of SNHG1 (Fig. 5E, F). Besides, the detection results of the TNF-α, IL-1β, IL-6, SOD, and MDA contents also showed that SNHG1 could invert the suppression function of Res on the inflammatory response and oxidative stress of SK-N-SH and CHP 212 induced by Aβ, while overexpressed miR-361-3p recovered the inhibition effects of SNHG1 on inflammatory response and oxidative stress (Fig. 5G–K). All data revealed that miR-361-3p played an important role in SNHG1 regulated cell injury.

Fig. 5

Effects of SNHG1 and miR-361-3p on Res and Aβ treated SK-N-SH and CHP 212 cells. A) The expression of miR-361-3p was assessed by qRT-PCR to evaluate the transfection efficiency of miR-361-3p mimic. SK-N-SH and CHP 212 cells were treated with 20μM Aβ and 20 nM Res and co-transfected with si-SNHG1#1 and miR-361-3p mimic. B) QRT-PCR was performed to test the miR-361-3p expression in SK-N-SH and CHP 212 cells. C) CCK8 assay was employed to detect the viability of SK-N-SH and CHP 212 cells. D) The apoptosis of SK-N-SH and CHP 212 cells was determined by flow cytometry. E, F) WB analysis was performed to measure the protein levels of Bax, Bcl-2, and Cleaved caspase 3/pro-caspase 3 in SK-N-SH and CHP 212 cells. G–K) The contents of TNF-α, IL-1β, IL-6, SOD, and MDA were tested by ELISA assay in SK-N-SH and CHP 212 cells. Student’s t-test was used for comparison in A, and two-way ANOVA followed by Tukey post hoc test was used for comparison in B-K. *p < 0.05.

MiR-361-3p directly targeted ZNF217

To search for the downstream targets of the SNHG1/miR-361-3p pathway, we used the ENCORI tool to make predictions, and the results showed that ZNF217 3’UTR had the binding sites of miR-361-3p (Fig. 6A). Dual-luciferase reporter assay results revealed that miR-361-3p overexpression markedly suppressed the luciferase activity of ZNF217-wt in SK-N-SH and CHP 212 cells, while had no effect on the luciferase activity of ZNF217-mut (Fig. 6B). RIP assay results showed that in SK-N-SH and CHP 212 cells, miR-361-3p and ZNF217 were significantly enriched in Anti-Ago2 (Fig. 6C). Moreover, we explored the effect of miR-361-3p expression on the ZNF217 protein level. The detection of miR-361-3p expression revealed that anti-miR-361-3p could effectively inhibit the expression of miR-361-3p, indicating successful transfection (Fig. 6D). Through measurement of the ZNF217 protein level, we discovered that ZNF217 expression was hindered by miR-361-3p overexpression, while enhanced by miR-361-3p inhibition in SK-N-SH and CHP 212 cells (Fig. 6E). These data suggested that ZNF217 was a target of miR-361-3p in SK-N-SH and CHP 212 cells.

Fig. 6

MiR-361-3p directly targeted ZNF217. A) The sequences of ZNF217 3’UTR containing the miR-361-3p binding sites or mutant binding sites were shown. B) The interaction between miR-361-3p and ZNF217 was assessed by dual-luciferase reporter assay in SK-N-SH and CHP 212 cells. C) RIP assay was used to measure the enrichment of miR-361-3p and ZNF217 in Anti-Ago2 or Anti-IgG. D) The expression of miR-361-3p was detected by qRT-PCR to evaluate the transfection efficiency of anti-miR-361-3p. E) WB analysis was performed to determine the protein level of ZNF217 in SK-N-SH and CHP 212 cells to evaluate the effect of miR-361-3p expression on ZNF217 expression. Student’s t-test was used for comparison. *p < 0.05.

Effects of miR-361-3p inhibitor and ZNF217 silencing on the treatment of Aβ and Res in SK-N-SH and CHP 212 cells

To confirm the role of ZNF217 on cell injury, we built si-ZNF217 and tested its transfection efficiency. WB analysis results indicated that si-ZNF217 could effectively restrain the expression of ZNF217 and could be used for the next study (Fig. 7A). Then, we co-transfected anti-miR-361-3p and si-ZNF217 into SK-N-SH and CHP 212 cells. Through the detection of ZNF217 expression, we found that Res could reverse the acceleration of Aβ on ZNF217 expression, and si-ZNF217 could invert the promotion effect of anti-miR-361-3p on ZNF217 expression (Fig. 7B). Besides, CCK8 assay results uncovered that miR-361-3p inhibitor inverted the promotion effect of Res on viability in Aβ-induced SK-N-SH and CHP 212 cells, while ZNF217 knockdown could recover this effect (Fig. 7C). Further, flow cytometry revealed that miR-361-3p inhibitor reversed the inhibition effect of Res on apoptosis in Aβ-induced SK-N-SH and CHP 212 cells, whereas this effect could be inverted by ZNF217 silencing, which could also be confirmed by the detection of apoptosis-related proteins (Fig. 7D–F). Meanwhile, miR-361-3p inhibitor could reverse the suppression function of Res on inflammatory responses and oxidative stress in Aβ-induced SK-N-SH and CHP 212 cells, while this effect could also be restored by ZNF217 knockdown (Fig. 7G–K). All data determined that ZNF217 was a key regulator of cell injury.

Fig. 7

Effects of miR-361-3p and ZNF217 on Res and Aβ treated SK-N-SH and CHP 212 cells. A) The protein level of ZNF217 was assessed by WB analysis to evaluate the transfection efficiency of si-ZNF217. SK-N-SH and CHP 212 cells were treated with 20μM Aβ and 20 nM Res and co-transfected with anti-miR-361-3p and si-ZNF217. B) WB analysis was performed to test the protein level of ZNF217 in SK-N-SH and CHP 212 cells. C) The viability of SK-N-SH and CHP 212 cells was tested by CCK8 assay. D) Flow cytometry was employed to measure the apoptosis of SK-N-SH and CHP 212 cells. E, F) The protein levels of Bax, Bcl-2, and Cleaved caspase 3/pro-caspase 3 in SK-N-SH and CHP 212 cells were detected by WB analysis. G–K) The contents of TNF-α, IL-1β, IL-6, SOD, and MDA in SK-N-SH and CHP 212 cells were determined by ELISA assay. Student’s t-test was used for comparison in A, and two-way ANOVA followed by Tukey post hoc test was used for comparison in B–K. *p < 0.05.

DISCUSSION

Currently, the deposition of Aβ is considered as the core etiology of AD, which can promote the development of AD by accelerating nerve necrosis, inflammation response, and oxidative stress [22]. A recent study showed that Res could inhibit oxidative stress in AD model mice and enhance their cognitive ability [23]. Also, the antioxidant and anti-inflammatory effects of Res had been proven to have a strong protective effect on the nerves of patients with AD [24, 25]. Therefore, Res might be a natural antioxidant that effectively alleviates the progression of AD. Here, we found that Aβ could hinder cell viability and promote cell apoptosis, inflammation response, and oxidative stress, suggesting that the use of Aβ-induced cell injury to construct an AD cell model in vitro was successful. Besides, Res could alleviate Aβ-induced cell injury, which was consistent with previous research results [23 –25].

The expression of lncRNAs has been confirmed to be closely related to the occurrence and development of neurodegenerative diseases [26]. Therefore, it is necessary to find effective lncRNAs to provide a theoretical basis for alleviating the progress of neurodegenerative diseases. Studies have confirmed that SNHG1 may promote neuroinflammation and neuronal cell apoptosis in Parkinson’s disease [16, 27]. Also, knockdown of SNHG1 can promote cell survival to alleviate the progression of AD [17]. Thus, SNHG1 may play a positive role in the progression of neurodegenerative diseases. In our study, we uncovered that SNHG1 was differentially expressed in the process of Aβ and Res regulating cell injury and recovery. Functional verification results showed that silenced SNHG1 could reverse the cell injury induced by Aβ, indicating that the downregulation of SNHG1 had a neuroprotective effect, which was similar to the function of Res. Additionally, overexpressed SNHG1 also inverted the recovery effect of Res on Aβ-induced cell injury. Consistent with the previous study [17], the neuroprotective effect of SNHG1 knockdown might provide a theoretical basis for the treatment of AD.

miRNA has been reported to play a key role in the progression of AD. Studies have shown that miR-153, miR-346, and miR-339-5p are dysregulated in the brain tissues of AD patients and regulate the progression of the AD by mediating the expression of amyloid-β protein precursor [28 –30]. It has been proven that many miRNAs can be adsorbed by lncRNAs and participate in the regulation of AD progression, such as miR-125b, miR-124, and miR-107 [11 , 31]. Herein, we discovered that SNHG1 could serve as a ceRNA for miR-361-3p. miR-361-3p could regulate Aβ accumulation to be involved in the regulation of cognitive deficits of AD [19]. Through functional tests, we proved that miR-361-3p overexpression could invert the cell injury induced by SNHG1 overexpression to increase cell viability and decrease cell apoptosis, inflammation response, and oxidative stress. miR-361-3p inhibitor also reversed the restorative function of Res on Aβ-induced cell injury. More importantly, further experiments verified that miR-361-3p could target ZNF217. Silenced ZNF217 could invert the effect of miR-361-3p inhibitor, which also confirmed that ZNF217 was an important regulator of AD progression and was consistent with previous conclusions [21].

At present, there is no effective treatment strategy for AD, and all drugs or treatment programs are designed to alleviate the progression of AD [32 –34]. An effective biomarker for AD may be helpful in developing new strategies for the treatment of AD. In this study, we found that the inhibitory effect of SNHG1 deletion on cell injury was similar to that of Res, suggesting that SNHG1 has the potential to be a new target to alleviate the progression of AD. Of course, there are some limitations to our research. Our study was only validated at the cellular level; the expression of SNHG1 in the brain tissues of AD patients and its role in vivo needed further verification.

Conclusion

In conclusion, our study revealed that SNHG1 could promote ZNF217 expression to regulate Aβ-induced cell injury through sponging miR-361-3p, which might provide help for the clinical treatment of AD patients.

DISCLOSURE STATEMENT

Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/19-1303r2).

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